CHL1 promotes Sema3A-induced growth cone collapse and neurite elaboration through a motif required for recruitment of ERM proteins to the plasma membrane

Close homolog of L1 (CHL1) is a transmembrane cell adhesion molecule with unique developmental functions in cortical neuronal positioning and dendritic projection within the L1 family, as well as shared functions in promotion of integrin-dependent neurite outgrowth and semaphorin3A (Sema3A)-mediated axon repulsion. The molecular mechanisms by which CHL1 mediates these diverse functions are obscure. Here it is demonstrated using a cytofluorescence assay that CHL1 is able to recruit ezrin, a member of the ezrin-radixin-moesin (ERM) family of filamentous actin binding proteins to the plasma membrane, and that this requires a membrane-proximal motif (RGGKYSV) in the CHL1 cytoplasmic domain. This sequence in CHL1 is shown to have novel functions necessary for Sema3A-induced growth cone collapse and CHL1-dependent neurite outgrowth and branching in cortical embryonic neurons. In addition, stimulation of haptotactic cell migration and cellular adhesion to fibronectin by CHL1 depends on the CHL1/ERM recruitment motif. These findings suggest that a direct or indirect interaction between CHL1 and ERM proteins mediates Sema3A-induced growth cone collapse as well as neurite outgrowth and branching, which are essential determinants of axon guidance and connectivity in cortical development.     

Neurotrophic effect of Semaphorin 4D in PC12 cells

Semaphorins provide crucial attractive and repulsive cues involved in axon guidance during neural development. Out of them, Semaphorin 4D (Sema4D) is enriched in the nervous and immune tissues, and acts as proliferative and survival factors of peripheral lymphocytes in the immune system, but is poorly understood in the nervous system. By using PC12 cells which are well known to differentiate into neural cells in response to nerve growth factor (NGF), we found that soluble forms of Sema4D had neurotrophic effects which were inhibited by neutralizing antibodies to Sema4D. Sema4D strikingly potentiated neurite outgrowth in the presence of 50 ng/ml NGF and increased sensitivity to NGF. Cells responded to very low concentrations of NGF in the presence of 1 nM Sema4D. Activation of following signal proteins, protein kinase C (PKC), L-type of voltage-dependent Ca(2+) channel, and phosphatidylinositol (PI) 3-kinase mediated neurotrophic neurite-outgrowth action of Sema4D. These findings suggest a new function of Sema4D as a neurotrophic signal in PC12 cells.     

Increasing tPA Activity in Astrocytes Induced by Multipotent Mesenchymal Stromal Cells Facilitate Neurite Outgrowth after Stroke in the Mouse

We demonstrate that tissue plasminogen activator (tPA) and its inhibitors contribute to neurite outgrowth in the central nervous system (CNS) after treatment of stroke with multipotent mesenchymal stromal cells (MSCs). In vivo, administration of MSCs to mice subjected to middle cerebral artery occlusion (MCAo) significantly increased activation of tPA and downregulated PAI-1 levels in the ischemic boundary zone (IBZ) compared with control PBS treated mice, concurrently with increases of myelinated axons and synaptophysin. In vitro, MSCs significantly increased tPA levels and concomitantly reduced plasminogen activator inhibitor 1 (PAI-1) expression in astrocytes under normal and oxygen and glucose deprivation (OGD) conditions. ELISA analysis of conditioned medium revealed that MSCs stimulated astrocytes to secrete tPA. When primary cortical neurons were cultured in the conditioned medium from MSC co-cultured astrocytes, these neurons exhibited a significant increase in neurite outgrowth compared to conditioned medium from astrocytes alone. Blockage of tPA with a neutralizing antibody or knock-down of tPA with siRNA significantly attenuated the effect of the conditioned medium on neurite outgrowth. Addition of recombinant human tPA into cortical neuronal cultures also substantially enhanced neurite outgrowth. Collectively, these in vivo and in vitro data suggest that the MSC mediated increased activation of tPA in astrocytes promotes neurite outgrowth after stroke.     

CRMP4 suppresses apical dendrite bifurcation of CA1 pyramidal neurons in the mouse hippocampus

Collapsin response mediator proteins (CRMPs) are a family of cytosolic phosphoproteins that consist of 5 members (CRMP 1–5). CRMP2 and CRMP4 regulate neurite outgrowth by binding to tubulin heterodimers, resulting in the assembly of microtubules. CRMP2 also mediates the growth cone collapse response to the repulsive guidance molecule semaphorin‐3A (Sema3A). However, the role of CRMP4 in Sema3A signaling and its function in the developing mouse brain remain unclear. We generated CRMP4?/? mice in order to study the in vivo function of CRMP4 and identified a phenotype of proximal bifurcation of apical dendrites in the CA1 pyramidal neurons of CRMP4?/? mice. We also observed increased dendritic branching in cultured CRMP4?/? hippocampal neurons as well as in cultured cortical neurons treated with CRMP4 shRNA. Sema3A induces extension and branching of the dendrites of hippocampal neurons; however, these inductions were compromised in the CRMP4?/? hippocampal neurons. These results suggest that CRMP4 suppresses apical dendrite bifurcation of CA1 pyramidal neurons in the mouse hippocampus and that this is partly dependent on Sema3A signaling. © 2012 Wiley Periodicals, Inc. Develop Neurobiol, 2012     

Elevation of Sema4A implicates Th cell skewing and the efficacy of IFN-β therapy in multiple sclerosis

Multiple sclerosis (MS) is a demyelinating autoimmune disease of the CNS and a leading cause of lasting neurologic disabilities in young adults. Although the precise mechanism remains incompletely understood, Ag presentation and subsequent myelin-reactive CD4(+) T cell activation/differentiation are essential for the pathogenesis of MS. Although semaphorins were initially identified as axon guidance cues during neural development, several semaphorins are crucially involved in various phases of immune responses. Sema4A is one of the membrane-type class IV semaphorins, which we originally identified from the cDNA library of dendritic cell (DC). Sema4A plays critical roles in T cell activation and Th1 differentiation during the course of experimental autoimmune encephalomyelitis, an animal model of MS; however, its pathological involvement in human MS has not been determined. In this study, we report that Sema4A is increased in the sera of patients with MS. The expression of Sema4A is increased on DCs in MS patients and shed from these cells in a metalloproteinase-dependent manner. DC-derived Sema4A is not only critical for Th1 but also for Th17 cell differentiation, and MS patients with high Sema4A levels exhibit Th17 skewing. Furthermore, patients with high Sema4A levels have more severe disabilities and are unresponsive to IFN-β treatment. Taken together, our results suggest that Sema4A is involved in the pathogenesis of MS by promoting Th17 skewing.     

Autocrine Semaphorin3A stimulates eukaryotic initiation factor 4E-dependent RhoA translation in breast tumor cells

Translation of the small G protein RhoA in neurons is regulated by the eukaryotic translation initiation factor eIF4E. Here we show that this translation factor also regulates RhoA expression and activity in breast cancer cells. The introduction of eIF4E into breast tumor cells increased RhoA protein levels, while expression of an eIF4E siRNA reduced RhoA expression. Previous studies indicate that the axon repulsion factor Semaphorin3A (Sema3A) stimulates the eIF4E-dependent translation of RhoA in neurons, and breast tumor cells support autocrine Sema3A signaling. Accordingly, we next examined if autocrine Sema3A signaling drives eIF4E-dependent RhoA translation in breast cancer cells. The incubation of breast tumor cells with recombinant Sema3A rapidly increased eIF4E activity, RhoA protein levels, and RhoA activity. This Sema3A activity was blocked in tumor cells expressing an shRNA-specific for the Sema3A receptor, Neuropilin-1 (NP-1), as well as in cells incubated with an eIF4E inhibitor. Importantly, RhoA protein levels were reduced in Sema3A shRNA-expressing compared to control shRNA-expressing breast tumor cells, demonstrating that autocrine Sema3A increases RhoA expression in breast cancer. Considering that Sema3A suppresses axon extension by stimulating RhoA translation, we next examined if the Sema3A/RhoA axis impacts breast tumor cell migration. The incubation of control breast tumor cells, but not RhoA shRNA-expressing cells, with rSema3A significantly reduced their migration. Collectively, these studies indicate that Sema3A impedes breast tumor cell migration in part by stimulating RhoA. These findings identify common signaling pathways that regulate the navigation of neurons and breast cancer cells, thus suggesting novel targets for suppressing breast tumor cell migration.     

Enhanced CCL26 production by IL-4 through IFN-gamma-induced upregulation of type 1 IL-4 receptor in keratinocytes

A Th2 cytokine, IL-4, induces various chemokines from epidermal keratinocytes which play crucial roles in the pathogenesis of skin disorders such as atopic dermatitis. In contrast, the role of IFN-γ, a Th1 cytokine, on eosinophilic skin inflammation is unclear. This study investigated the effects of IFN-γ on IL-4-induced production of eotaxin-3/CCL26, a potent chemoattractant to eosinophils, in normal human epidermal keratinocytes (NHEK). When the cells were stimulated with IL-4 and IFN-γ simultaneously, IL-4-induced CCL26 production was attenuated. In contrast, prior stimulation with IFN-γ enhanced IL-4-induced CCL26 production. NHEK constitutively expressed type 1 IL-4 receptor, and expression at the cell surface was upregulated by stimulation with IFN-γ. This upregulation resulted in an enhanced IL-4-mediated cellular signal. These results indicate that IFN-γ has opposite effects on IL-4-induced CCL26 production in NHEK depending on the time of exposure. Thus, changes in IL-4R expression by IFN-γ might modulate eosinophilic skin inflammation.     

Neurite outgrowth from progeny of epidermal growth factor–responsive hippocampal stem cells is significantly less robust than from fetal hippocampal cells following grafting onto organotypic hippocampal slice cultures: Effect of brain‐derived neurotrophic factor

Epidermal growth factor (EGF)–responsive stem cells from both developing and adult central nervous system (CNS) can be expanded and induced to differentiate into neurons and glia in vitro. Because of their self‐renewal and multipotent properties, these cells can potentially provide an unlimited tissue source for neural grafting in neurodegenerative disorders. However, the capability of neurons derived from these stem cells to project axons to distant targets following grafting, thereby enabling the restoration of damaged CNS circuitry, remains unknown. We hypothesize that grafted EGF‐responsive stem cells and their progeny are not competent to project axons into distant target sites unless exposed to specific neurotrophic factors. We compared neurite outgrowth between gestation day 14 primary mouse hippocampal cells and EGF‐generated secondary neurospheres of postnatal mouse hippocampal stem cells, following grafting onto the CA3 region of organotypic hippocampal slice cultures prepared from postnatal rats. Neurite outgrowth from grafted cells was visualized using immunohistochemical staining for the mouse specific antigen M6. Fetal hippocampal cells showed extensive and specific neurite outgrowth into many regions of the slice, including the CA1 region and distant subiculum, by 7 days after grafting. In contrast, neurite outgrowth from neurosphere cells was nonspecific and restricted to the immediate surrounding region after either 7 or even 15 days following grafting. Application of brain‐derived neurotrophic factor (BDNF) (5 ng in 0.5 μL) to slices on day 1 after grafting significantly enhanced neurite outgrowth from neurosphere cells, but overall neurite outgrowth from neurosphere cells remained decreased compared to that from fetal hippocampal cells. These results underscore that EGF‐responsive stem cell‐derived neurons possess limited intrinsic capability for long‐distance neurite outgrowth compared to fetal neurons. However, neurite outgrowth from EGF‐responsive stem cell–derived neurons can be enhanced by treating with specific neurotrophic factors such as BDNF. © 1999 John Wiley & Sons, Inc. J Neurobiol 38: 391–413, 1999     

Maturation of astrocytes in vitro alters the extent and molecular basis of neurite outgrowth

In the developing mammalian central nervous system astrocytes have been proposed as an important substrate for axon growth. In the adult central nervous system following injury, astrocytes are a major component of the gliotic response which has been proposed to block axon growth. Experimental transplantation studies using cultured astrocytes have suggested that immature but not mature cultured astrocytes have the capacity to support axon outgrowth when transplanted into the adult rodent CNS. These observations suggest that astrocyte maturation is accompanied by changes in the functional capacity of these cells to support axon outgrowth. To determine whether this functional change reflects an intrisic astrocyte property, the extent and molecular bases of neurite outgrowth from embryonic rat cortical and chick retinal neurons on cultures of purified immature and mature astrocytes have been compared in vitro. The rate and extent of neurite outgrowth from both neuronal populations are consistently greater over the surface of immature than over the surface of mature astrocytes. Furthermore, antibodies to NCAM and G4/L1 significantly reduce neurite outgrowth on immature but not mature astrocytes, while antibodies to the integrin B1 receptor reduced outgrowth on both immature and, to a lesser extent, mature astrocytes. These results suggest that in vitro mature astrocytes have a reduced capacity and different molecular bases for supporting neurite outgrowth compared to immature astrocytes and are consistent with the proposal that functional changes during astrocyte maturation may partially contribute to regulating axon growth in the mammalian CNS.     

Uncarinic acid C plus IFN-γ generates monocyte-derived dendritic cells and induces a potent Th1 polarization with capacity to migrate

Uncarinic acid C (URC) is triterpene isolated from Uncaria rhynchophylla and modulates human DC function in a fashion that favors Th1 cell polarization depending on TLR4 signaling. The induction of dendritic cells (DC) is critical for the induction of Ag-specific T lymphocyte responses and may be essential for the development of human vaccines relying on T cell immunity. Monocyte-derived DC used as adjuvant cells in cancer immunotherapy and have shown promising results. We studied the effect of interferon’s (IFN-α and IFN-γ) and TNF-α on phenotypic and functional maturation, and cytokine production of URC-primed DC in vitro. Human monocytes were exposed to either URC alone, or in combination with TNF-α, IFN-α or IFN-γ, and thereafter co-cultured with naïve T cells. We found that the expression levels of CD1a, CD83 and HLA-DR on URC-primed DC were influenced by IFN-γ and IFN-γ augmented the T cell stimulatory capacity in allo MLR to URC-primed DC. Moreover, the production of IL-12p70 by URC-primed DC was enhanced by IFN-γ. IL-12p70 production by URC-primed DC alone was influenced following treatment with anti-TLR4 mAb, but not DC differentiated with URC plus IFN-γ. URC plus IFN-γ-primed DC induced a substantial increase in the secretion of IFN-γ by T cells, which is dependent on IL-12 secretion. DC maturated with URC plus IFN-γ had an intermediate migratory capacity towards CCL19 and CCL21. In addition, the expression levels of CCR7 on URC-primed DC were enhanced by IFN-γ. In contrast, surface molecule up-regulation and function of URC-primed DC were slightly enhanced by TNF-α, and IFN-α. These results suggest that the enhancement of Th1 cells polarization to URC-primed DC induced by IFN-γ depends on the activation of IL-12p70 and independent on TLR4. DC differentiated with URC in combination with IFN-γ might be used on DC-based vaccine for cancer immunotherapy.     

MicroRNA miR-124 regulates neurite outgrowth during neuronal differentiation

MicroRNAs (miRNAs) are small RNAs with diverse regulatory roles. The miR-124 miRNA is expressed in neurons in the developing and adult nervous system. Here we show that overexpression of miR-124 in differentiating mouse P19 cells promotes neurite outgrowth, while blocking miR-124 function delays neurite outgrowth and decreases acetylated α-tubulin. Altered neurite outgrowth also was observed in mouse primary cortical neurons when miR-124 expression was increased, or when miR-124 function was blocked. In uncommitted P19 cells, miR-124 expression led to disruption of actin filaments and stabilization of microtubules. Expression of miR-124 also decreased Cdc42 protein and affected the subcellular localization of Rac1, suggesting that miR-124 may act in part via alterations to members of the Rho GTPase family. Furthermore, constitutively active Cdc42 or Rac1 attenuated neurite outgrowth promoted by miR-124. To obtain a broader perspective, we identified mRNAs downregulated by miR-124 in P19 cells using microarrays. mRNAs for proteins involved in cytoskeletal regulation were enriched among mRNAs downregulated by miR-124. A miR-124 variant with an additional 5′ base failed to promote neurite outgrowth and downregulated substantially different mRNAs. These results indicate that miR-124 contributes to the control of neurite outgrowth during neuronal differentiation, possibly by regulation of the cytoskeleton.     

Cholesterol-mediated neurite outgrowth is differently regulated between cortical and hippocampal neurons

The acquisition of neuronal type-specific morphogenesis is a central feature of neuronal differentiation and has important consequences for region-specific nervous system functions. Here, we report that the cell type-specific cholesterol profile determines the differential modulation of axon and dendrite outgrowths in hippocampal and cerebral cortical neurons in culture. The extent of axon and dendrite outgrowths is greater and the polarity formation occurs earlier in cortical neurons than in hippocampal neurons. The cholesterol concentrations in total homogenate and the lipid rafts from hippocampal neurons are significantly higher than those from cortical neurons. Cholesterol depletion by beta-cyclodextrin markedly enhanced the neurite outgrowth and accelerated the establishment of neuronal polarity in hippocampal neurons, which were similarly observed in nontreated cortical neurons, whereas cholesterol loading had no effects. In contrast, both depletion and loading of cholesterol decreased the neurite outgrowths in cortical neurons. The stimulation of neurite outgrowth and polarity formation induced by cholesterol depletion was accompanied by an enhanced localization of Fyn, a Src kinase, in the lipid rafts of hippocampal neurons. A concomitant treatment with beta-cyclodextrin and a Src family kinase inhibitor, PP2, specifically blocked axon outgrowth but not dendrite outgrowth (both of which were enhanced by beta-cyclodextrin) in hippocampal neurons, suggesting that axon outgrowth modulated by cholesterol is induced in a Fyn-dependent manner. These results suggest that cellular cholesterol modulates axon and dendrite outgrowths and neuronal polarization under culture conditions and also that the difference in cholesterol profile between hippocampal and cortical neurons underlies the difference in neurite outgrowth between these two types of neurons.     

Neurite outgrowth from progeny of epidermal growth factor-responsive hippocampal stem cells is significantly less robust than from fetal hippocampal cells following grafting onto organotypic hippocampal slice cultures: effect of brain-derived neurotrophic factor

Epidermal growth factor (EGF)-responsive stem cells from both developing and adult central nervous system (CNS) can be expanded and induced to differentiate into neurons and glia in vitro. Because of their self-renewal and multipotent properties, these cells can potentially provide an unlimited tissue source for neural grafting in neurodegenerative disorders. However, the capability of neurons derived from these stem cells to project axons to distant targets following grafting, thereby enabling the restoration of damaged CNS circuitry, remains unknown. We hypothesize that grafted EGF-responsive stem cells and their progeny are not competent to project axons into distant target sites unless exposed to specific neurotrophic factors. We compared neurite outgrowth between gestation day 14 primary mouse hippocampal cells and EGF-generated secondary neurospheres of postnatal mouse hippocampal stem cells, following grafting onto the CA3 region of organotypic hippocampal slice cultures prepared from postnatal rats. Neurite outgrowth from grafted cells was visualized using immunohistochemical staining for the mouse specific antigen M6. Fetal hippocampal cells showed extensive and specific neurite outgrowth into many regions of the slice, including the CA1 region and distant subiculum, by 7 days after grafting. In contrast, neurite outgrowth from neurosphere cells was nonspecific and restricted to the immediate surrounding region after either 7 or even 15 days following grafting. Application of brain-derived neurotrophic factor (BDNF) (5 ng in 0.5 microL) to slices on day 1 after grafting significantly enhanced neurite outgrowth from neurosphere cells, but overall neurite outgrowth from neurosphere cells remained decreased compared to that from fetal hippocampal cells. These results underscore that EGF-responsive stem cell-derived neurons possess limited intrinsic capability for long-distance neurite outgrowth compared to fetal neurons. However, neurite outgrowth from EGF-responsive stem cell-derived neurons can be enhanced by treating with specific neurotrophic factors such as BDNF.     

The role of gamma interferon in DNA vaccine-induced tumor immunity targeting simian virus 40 large tumor antigen

The central role of CD4+ T lymphocytes in mediating DNA vaccine-induced tumor immunity against the viral oncoprotein simian virus 40 (SV40) large tumor antigen (Tag) has previously been described by our laboratory. In the present study, we extend our previous findings by examining the roles of IFN-γ and Th1-associated effector cells within the context of DNA immunization in a murine model of pulmonary metastasis. Immunization of BALB/c mice with plasmid DNA encoding SV40 Tag (pCMV-Tag) generated IFN-γ-secreting T lymphocytes that produced this cytokine upon in vitro stimulation with mKSA tumor cells. The role of IFN-γ as a mediator of protection against mKSA tumor development was assessed via in vivo IFN-γ neutralization, and these experiments demonstrated a requirement for this cytokine in the induction immune phase. Neutralization of IFN-γ was associated with a reduction in Th1 cytokine-producing CD4+ and CD8+ splenocytes, as assessed by flow cytometry analysis, and provided further evidence for the role of CD4+ T lymphocytes as drivers of the cellular immune response. Depletion of NK cells and CD8+ T lymphocytes demonstrated the expendability of these cell types individually, but showed a requirement for a resident cytotoxic cell population within the immune effector phase. Our findings demonstrate the importance of IFN-γ in the induction of protective immunity stimulated by pCMV-Tag DNA-based vaccine and help to clarify the general mechanisms by which DNA vaccines trigger immunity to tumor cells.     

Mitochondrial adenine nucleotide translocator 3 is regulated by IL-4 and IFN-gamma via STAT-dependent pathways

IL-4 and IFN-γ are prototypical Th2 and Th1 cytokines, respectively. They reciprocally regulate a number of genes involved in Th1 vs Th2 immune balance. Using DD-PCR analysis, adenine nucleotide translocase (ANT) 3, an enzyme which exchanges ATP and ADP through mitochondrial membrane, has been identified as a novel target counter-regulated by IL-4 and IFN-γ. We have observed that IL-4 and IFN-γ each up-regulates ANT3 in T cells both at mRNA and protein levels, while cotreatment of IL-4 and IFN-γ counter-regulates ANT3 expression. In contrast, other isoforms of ANT were not affected by IL-4 or IFN-γ. Emplyoing transfection and overexpression of STAT6 and STAT1 in STAT-deficient cells, we demonstrate that induction of ANT3 by IL-4 and IFN-γ proceeds via pathways involving STAT6 and STAT1, respectively. Furthermore, regulation of ANT3 expression by IL-4 and IFN-γ correlated with the modulation T cell survival by these cytokines from dex-induced apoptosis. Considering the critical role of mitochondrial ANTs in energy metabolism and apoptosis, ANT3 regulation by IL-4 and IFN-γ may have a functional implication in cytokine-mediated T cell survival.     

The Promotive Effects of Thymosin β4 on Neuronal Survival and Neurite Outgrowth by Upregulating L1 Expression

Thymosin β₄ (Tβ4) is a major actin-sequestering peptide widely distributed in mammalian tissues including the nervous system. The presence of this peptide in the nervous system likely plays a role in synaptogensis, axon growth, cell migration, and plastic changes in dendritic spine. However, the effects of Tβ4 on the survival of neurons and axonal outgrowth have still not been fully understood. So far it is not clear if the effects of Tβ4 are associated with L1 functions. In the present study, we hypothesized that Tβ4-induced up-regulation of L1 synthesis could be involved in the survival and axon outgrowth of cultured spinal cord neurons. To test this hypothesis, primarily cultured neurons were prepared from the mouse spinal cord and treated with various concentrations of Tβ4 ranging from 0.1 to 10 μg/ml. The analysis of L1 mRNA expression and protein synthesis in neurons was then carried out using RT-PCR and western blot assays, respectively. After the addition of Tβ4 to cultures, cells were then treated with antibodies against distinct domains of L1-Fc. Subsequently, β-tubulin III and L1 double-labeled indirect immunofluorescence was carried out. Meanwhile, L1 immunofluorescent reactivity was analyzed and compared in cells treated with Tβ4. Furthermore, the number of β-tubulin III-positive cells and neurite lengths were measured. We found that Tβ4 enhanced L1 expression in a dose-dependent manner, and the highest L1 mRNA and protein synthesis in cells increased by more than 2.1- and 2.3-fold in the presence of Tβ4 at identical concentrations, respectively. Moreover, it also dose dependently enhanced neurite outgrowth and neuronal survival. Compared to conditions without Tβ4, the length of neurite and neuronal survival increased markedly in presence of 0.5, 1, and 5 μg/ml Tβ4, respectively, whereas the effects of Tβ4 were significantly attenuated or inhibited in the process of L1-Fc antibodies treatment. These above results indicate that the promotive effect of Tβ4 on the survival and neurite outgrowth of cultured spinal cord neurons might be mediated, at least in part via a stimulation of the production of L1 in the neurons.     

VEGF-A and Semaphorin3A: Modulators of vascular sympathetic innervation

Sympathetic nerve activity regulates blood pressure by altering peripheral vascular resistance. Variations in vascular sympathetic innervation suggest that vascular-derived cues promote selective innervation of particular vessels during development. As axons extend towards peripheral targets, they migrate along arterial networks following gradients of guidance cues. Collective ratios of these gradients may determine whether axons grow towards and innervate vessels or continue past non-innervated vessels towards peripheral targets. Utilizing directed neurite outgrowth in a three-dimensional (3D) co-culture, we observed increased axon growth from superior cervical ganglion explants (SCG) towards innervated compared to non-innervated vessels, mediated in part by vascular endothelial growth factor (VEGF-A) and Semaphorin3A (Sema3A) which both signal via neuropilin-1 (Nrp1). Exogenous VEGF-A, delivered by high-expressing VEGF-A-LacZ vessels or by rhVEGF-A/alginate spheres, increased sympathetic neurite outgrowth while exogenous rhSema3A/Fc decreased neurite outgrowth. VEGF-A expression is similar between the innervated and non-innervated vessels examined. Sema3A expression is higher in non-innervated vessels. Spatial gradients of Sema3A and VEGF-A may promote differential Nrp1 binding. Vessels expressing high levels of Sema3A favor Nrp1-PlexinA1 signaling, producing chemorepulsive cues limiting sympathetic neurite outgrowth and vascular innervation; while low Sema3A expressing vessels favor Nrp1-VEGFR2 signaling providing chemoattractive cues for sympathetic neurite outgrowth and vascular innervation.     

Outer membrane protein A (OmpA) of Shigella flexneri 2a links innate and adaptive immunity in a TLR2-dependent manner and involvement of IL-12 and nitric oxide

We determine that OmpA of Shigella flexneri 2a is recognized by TLR2 and consequently mediates the release of proinflammatory cytokines and activates NF-κB in HEK 293 cells transfected with TLR2. We also observe that in RAW macrophages TLR2 is essential to instigate the early immune response to OmpA via NF-κB activation and secretion of cytokines and NO. Consistent with these results, TLR2 knockdown using siRNA abolishes the initiation of immune responses. Processing and presentation of OmpA depend on TLR2; MHCII presentation of the processed antigen and expression of CD80 significantly attenuated in TLR2 knockdown macrophages. The optimum production of IFN-γ by the macrophages:CD4(+) T cells co-culture depends on both TLR2 activation and antigen presentation. So, TLR2 is clearly recognized as a decisive factor in initiating host innate immune response to OmpA for the development of CD4(+) T cell adaptive response. Furthermore, we demonstrate in vivo that intranasal immunization of mice with OmpA selectively enhances the release of IFN-γ and IL-2 by CD4(+) T cells. Importantly, OmpA increases the level of IFN-γ production in Ag-primed splenocytes. The addition of neutralizing anti-IL-12p70 mAb to cell cultures results in the decreased release of OmpA-enhanced IFN-γ by Ag-primed splenocytes. Moreover, coincubation with OmpA-pretreated macrophages enhances the production of IFN-γ by OmpA-primed CD4(+) T cells, representing that OmpA may enhance IFN-γ expression in CD4(+) T cells through the induction of IL-12 production in macrophages. These results demonstrate that S. flexneri 2a OmpA may play a critical role in the development of Th1 skewed adaptive immune response.